Presently, many effective methods for the identification of microorganisms are available. These methods vary widely in sensitivity and specificity. Most commonly, cultural examinations of bacteria are made which require much time and often are not specific unless extremely tedious procedures are followed. The lack of speed characteristic of many routine analyses is a serious problem. Frequently, important decisions relating to the presence of pathogens have to be made before the results of microbiological tests are available, because present methods of detection and identification are, in many cases, too slow or nonspecific to be of much immediate help to decision-makers or potential victims.
The last quarter century, and especially the last decade, has seen a revolution in the application of sensitive and rapid methods of chemical analysis. This has happened, to a large extent, due to advances in electronics, optics, and computer technology which have allowed the practical application of physical methods which previously had been understood in theory, but were too cumbersome to use. These methods have had a major impact on analytical laboratories by making previously difficult analyses affordable and routine and by providing many opportunities for automation.
However, until relatively recently, there was little promise of applying these sophisticated new techniques to biodetection because of the lack of information regarding the molecular composition of microorganisms. Today, chemical information can be used effectively to establish relationships at all levels in the taxonomic hierarchy. Chemical properties, it appears, can and must be used in description of many genera and species.
The progress of biochemists and microbiologists in characterizing and identifying chemical markers has not gone unnoticed by chemical analysts. During the past several years, there has been marked progress in methods of chemical analysis and automation in biodetection and identification. Several potentially rapid new physical methods have been developed in the past several years which promise to achieve truly rapid analysis.
Among the most highly developed of the new rapid techniques is mass spectroscopy and its various combinations with gas chromatography (bacterial biproducts from cultures) and pyrolysis methods. Gas chromatography is highly effective in detecting characteristic bacterial metabolic products. Flow cytometry has provided means for the rapid detection, identification, and separation of cells. Total luminescence spectroscopy can detect organisms very rapidly. The various immunological methods also can be very specific and very rapid. All of these methods have their distinct advantages and disadvantages.
Mass spectroscopy may be unequalled in identification of pure cultures, and it is very rapid and sensitive. However, it is expensive to use, requires the destruction of samples, and is of questionable use in the analysis of complex mixtures. Flow cytometry is perhaps even more costly, requires extensive sample preparation, and in many aspects is limited in its scope of applicability. Luminescence techniques are of little use except in studies of pure cultures unless combined with immunological methods. Immunological methods are unequalled in specificity and speed, as well as sensitivity. Yet, they are often impractical to use unless very expensive and perishable materials are available in a state of constant readiness. Such methods are not practical for a wide range of organisms. Gas chromatography requires that cells be grown and, hence, this method is generally slow and of limited applicability.
The present invention is directed to a new method, and a system embodying that method, for the rapid detection and identification of bacteria and other microorganisms. The invention broadly includes a method wherein a beam of visible or ultraviolet light energy contacts a microorganism under investigation. A portion of the light energy is absorbed by the microorganism and a portion of the light energy is `emitted` from the sample at a lower energy level. The emitted light energy (resonance enhanced Raman scattering) may be measured at any angle but preferably is measured as backscattered energy. This energy is processed to produce spectra which are inherently characteristic of the microorganism.
The light energy which contacts the microorganism may be at any wavelenth so long as it corresponds to a molecular electronic transition which corresponds to strong absorption by the organism. Preferably the energy is a single selected wavelength in the ultraviolet range since most electronic transitions of component molecules of microorganisms occur in that range.
In a preferred embodiment, the emitted energy measured is based upon ultraviolet resonance Raman spectroscopy. Bacteria under investigation are struck by an incident beam of light energy, typically a single wavelength in the ultraviolet range. The emitted energy is collected, collimated and focused onto the entrance slit of a monochromator. The beam strikes a grating or gratings and the wavelengths reflected by the grating or gratings are plotted versus intensity to obtain a spectrum. The chemotaxonomic markers inherent in the bacteria are different for each bacterial type and these differences are reflected in the distinct spectra generated. With a known sample the characteristic spectra are plotted and these spectra, which are stored, are the `fingerprints` of that bacterium. When unknown samples are analyzed, their spectra are compared with the known spectra in memory to determine the identity of the unknown sample.